mRNA-based VP8* nanoparticle vaccines against rotavirus are highly immunogenic in rodents

Despite the availability of live-attenuated oral vaccines, rotavirus remains a major cause of severe childhood diarrhea worldwide. Due to the growing demand for parenteral rotavirus vaccines, we developed mRNA-based vaccine candidates targeting the viral spike protein VP8*. Our monomeric P2 (universal T cell epitope)-VP8* mRNA design is equivalent to a protein vaccine currently in clinical development, while LS (lumazine synthase)-P2-VP8* was designed to form nanoparticles. Cyro-electron microscopy and western blotting-based data presented here suggest that proteins derived from LS-P2-VP8* mRNA are secreted in vitro and self-assemble into 60-mer nanoparticles displaying VP8*. mRNA encoded VP8* was immunogenic in rodents and introduced both humoral and cellular responses. LS-P2-VP8* induced superior humoral responses to P2-VP8* in guinea pigs, both as monovalent and trivalent vaccines, with encouraging responses detected against the most prevalent P genotypes. Overall, our data provide evidence that trivalent LS-P2-VP8* represents a promising mRNA-based next-generation rotavirus vaccine candidate.

1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure: ELECTRON MICROSCOPY The reported resolution of this entry is 2.60 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic.The table shows the number of entries on which the scores are based.The table below summarises the geometric issues observed across the polymeric chains and their fit to the map.The red, orange, yellow and green segments of the bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively.A grey segment represents the fraction of residues that are not modelled.The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5%

Metric
The upper red bar (where present) indicates the fraction of residues that have poor fit to the EM map (all-atom inclusion < 40%).The numeric value is given above the bar.
Mol Chain Length Quality of chain  2 Entry composition i ○ There is only 1 type of molecule in this entry.The entry contains 70560 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called 6,7-dimethyl-8-ribityllumazine synthase.There are 60 discrepancies between the modelled and reference sequences: F o r

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Page 11 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-28807, 8F25 3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry.The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and atom inclusion in map density.Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more.A red diamond above a residue indicates a poor fit to the EM map for this residue (all-atom inclusion < 40%).Stretches of 2 or more consecutive residues without any outlier are shown as a green connector.Residues present in the sample, but not in the model, are shown in grey.

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Page 20 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-28807, 8F25 Chain w: • Molecule 1: 6,7-dimethyl-8-ribityllumazine synthase Chain x: There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
Due to software issues we are unable to calculate clashes -this section is therefore empty.

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Page 24 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-28807, 8F25

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all PDB entries followed by that with respect to all EM entries.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.There are no Ramachandran outliers to report.

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all PDB entries followed by that with respect to all EM entries.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.6 Map visualisation i ○ This section contains visualisations of the EMDB entry EMD-28807.These allow visual inspection of the internal detail of the map and identification of artifacts.
Images derived from a raw map, generated by summing the deposited half-maps, are presented below the corresponding image components of the primary map to allow further visual inspection and comparison with those of the primary map.

Orthogonal projections
The images above show the map projected in three orthogonal directions.The images above show central slices of the map in three orthogonal directions.

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Page 32 Full wwPDB EM Validation Report (*For Manuscript Review*) EMD-28807, 8F25 The images above show the largest variance slices of the map in three orthogonal directions.The images above show the 3D surface view of the map at the recommended contour level 4.0.These images, in conjunction with the slice images, may facilitate assessment of whether an appropriate contour level has been provided.

X Y Z
These images show the 3D surface of the raw map.The raw map's contour level was selected so that its surface encloses the same volume as the primary map does at its recommended contour level.

Mask visualisation i ○
This section was not generated.No masks/segmentation were deposited.7 Map analysis i ○ This section contains the results of statistical analysis of the map.

Map-value distribution i ○
The map-value distribution is plotted in 128 intervals along the x-axis.The y-axis is logarithmic.A spike in this graph at zero usually indicates that the volume has been masked.The images above show the model with each residue coloured according its Q-score.This shows their resolvability in the map with higher Q-score values reflecting better resolvability.Please note: Q-score is calculating the resolvability of atoms, and thus high values are only expected at resolutions at which atoms can be resolved.Low Q-score values may therefore be expected for many entries.

Atom inclusion i ○
At the recommended contour level, 94% of all backbone atoms, 83% of all non-hydrogen atoms, are inside the map.
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estimate i ○The volume at the recommended contour level is 366 nm 3 ; this corresponds to an approximate mass of 331 kDa.The volume estimate graph shows how the enclosed volume varies with the contour level.The recommended contour level is shown as a vertical line and the intersection between the line and the curve gives the volume of the enclosed surface at the given level.averaged power spectrum i ○ *Reported resolution corresponds to spatial frequency of 0.385 Å −1 Correlation (FSC) is the most commonly used method to estimate the resolution of single-particle and subtomogram-averaged maps.The shape of the curve depends on the imposed symmetry, mask and whether or not the two 3D reconstructions used were processed from a common reference.The reported resolution is shown as a black line.A curve is displayed for the half-bit criterion in addition to lines showing the 0.143 gold standard cut-off and 0.5 cut-off.8.1 FSC i ○ *Reported resolution corresponds to spatial frequency of 0.385 Å −1

9
Map-model fit i ○This section contains information regarding the fit between EMDB map EMD-28807 and PDB model 8F25.Per-residue inclusion information can be found in section 3 on page 11.9.1 Map-model overlayi ○ X Y ZThe images above show the 3D surface view of the map at the recommended contour level 4.0 at 50% transparency in yellow overlaid with a ribbon representation of the model coloured in blue.These images allow for the visual assessment of the quality of fit between the atomic model and the map.

9. 3
Atom inclusion mapped to coordinate modeli ○ X Y ZThe images above show the model with each residue coloured according to its atom inclusion.This shows to what extent they are inside the map at the recommended contour level (4.0).

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8.2 Resolution estimates i○ Resolution estimate based on FSC curve calculated by comparison of deposited half-maps.The value from deposited half-maps intersecting FSC 0.143 CUT-OFF 3.05 differs from the reported value 2.6 by more than 10 % *